Osteoblasts mineralize the pericellular matrix by promoting initial formation of crystalline hydroxyapatite in the sheltered interior of membrane-limited matrix vesicles (MVs) and by modulating matrix composition to further promote propagation of apatite outside of the MVs. Three molecules present in osteoblasts have been identified by means of gene knock-out models as affecting the controlled deposition of bone mineral, i.e., alkaline phosphatase (TNAP); PC-1 (or Npps, a nucleoside triphosphate pyrophosphate hydrolase isozyme, NTPPPH) and ANK, a multipass membrane protein that appears to serve as an anion channel. Our inactivation of the mouse TNAP gene led to the development of a model of Infantile Hypophosphatasia characterized by undermineralization of bone (osteomalacia). Inactivation of the PC-1 gene causes systemic hyperossification and skeletal and extraskeletal apatite deposition. Moreover, ANK-deficient ank/ank mutant mice have recently been described as developing a phenotype remarkably similar to that of the PC-1 null mice.
A deficiency in the TNAP isozyme causes hypophosphatasia, the study of which has provided the best evidence of the importance of TNAP for bone mineralization. TNAP is the only tissue-nonrestricted isozyme of a family of four homologous human. AP genes (EC. 3.1.3.1) (Millán and Fishman, Crit. Rev. Clin. Lab. Sci. 32:1-39, 1995). Expressed as an ecto-enzyme transported to the osteoblast plasma membrane and anchored via a phosphatidylinositol glycan moiety, TNAP has been demonstrated to play an essential physiological role during osteoblastic bone matrix mineralization (Whyte, Endocrine Rev. 15:439-461, 1994; Narisawa et al., Dev. Dynamics 208:432-446, 1997; Zurutuza et al., Hum. Mol. Genet. 8:1039-1046, 1999). Specifically, defective bone mineralization (osteomalacia) occurs in TNAP deficiency (hypophosphatasia) (Whyte, Endocrine Rev. 15:439-461, 1994). The severity of hypophosphatasia is variable and modulated by the nature of the TNAP mutation (Henthorn et al., Proc. Nat. Acad. Sci. U.S.A. 89:9924-9928, 1992; Fukushi et al., Biochem. Biophys. Res. Comm. 246:613-618, 1998; Shibata et al., J. Biochem. 123:968-977, 1998; Narisawa et al., Dev. Dynamics 208:432-446, 1997; Zurutuza et al., Hum. Mol. Genet. 8:1039-1046, 1999; Di Mauro et al., J. Bone Min. Res. 17: 1383-1391, 2002). Unlike most types of rickets or osteomalacia, neither calcium nor inorganic phosphate levels in serum are subnormal in hypophosphatasia. In fact hypercalcemia and hyperphosphatemia may exist, and hypercalciuria is common in infantile hypophosphatasia (Whyte, “Hypophosphatasia,” In: The Metabolic and Molecular Bases of Inherited Disease, ed. Scriver et al., McGraw-Hill Inc., New York, pp. 4095-4112, 1995). The clinical severity in hypophosphatasia patients varies widely. The different syndromes, listed from the most severe to the mildest forms, are: perinatal hypophosphatasia, infantile hypophosphatasia, childhood hypophosphatasia, adult hypophosphatasia, odontohypophosphatasia and pseudohypophosphatasia (Whyte, “Hypophosphatasia,” In: The Metabolic and Molecular Bases of Inherited Disease, ed. Scriver et al., McGraw-Hill Inc., New York, pp. 4095-4112, 1995). These phenotypes range from complete absence of bone mineralization and stillbirth to spontaneous fractures and loss of decidual teeth in adult life.
Physiologic bone matrix mineralization is hypothesized to be dependent on the availability of Pi released from a variety of substrates by certain MV ecto-enzymes (Anderson, Clin. Orthopaed. Rel. Res. 314:266-280, 1995; Hsu and Anderson, J. Biol. Chem. 271:26383-26388, 1996). For example, ATP is hypothesized to drive the initiation of calcification by MVs in vivo, and a specific bone and cartilage ATPase appears to be responsible for the ATP-dependent calcium and Pi-depositing activity of bone and cartilage-derived MVs in vitro (Hsu and Anderson, 1996; Pizauro et al., 1998). Skeletal TNAP can catalyze Pi release from ATP (Hsu and Anderson, J. Biol. Chem. 271:26383-26388, 1996; Pizauro et al., Biochim. Biophys. Acta 1368:108-114, 1997), TNAP catalyzes several transphosphorylation reactions (Whyte, Endocrine Rev. 15:439-461, 1994) and TNAP can also function as a pyrophosphatase (Moss et al., Biochem. J. 102:53-57, 1967; Rezende et al., Biochem. J. 301:517-522, 1994). Although TNAP does not appear to dephosphorylate membrane proteins (Fedde et al., J. Cell. Biochem. 53:43-50, 1993), TNAP has been hypothesized to modulate bridging of MVs to matrix collagen (Whyte, Endocrine Rev. 15:439-461, 1994; Henthom et al., “Acid and alkaline phosphatases,” In: Principles of Bone Biology, eds. Seibel et al., Academic Press, pp. 127-137, 1999). TNAP has been demonstrated to bind calcium (de Bernard et al., J. Cell. Biol. 103:1615-1623, 1986). Moreover, TNAP degrades at least three phosphocompounds, i.e., phosphoethanolamine, pyridoxal 5′ phosphate, and PPi, that accumulate endogenously in hypophosphatasia (Whyte et al., “Hypophosphatasia,” In: The Metabolic and Molecular Bases of Inherited Disease, ed. Scriver et al., McGraw-Hill Inc., New York, pp. 4095-4112, 1995). The central function or functions of TNAP in conditioning mineralization have not been completely defined (Whyte, Endocrine Rev. 15:439-461, 1994). Importantly, aberrant localization of TNAP can occur, including defective transport of TNAP to the plasma membrane associated with hypophosphatasia (Fedde et al., Am. J. Hum. Genet. 47: 767-775, 1990; Fedde et al., Am. J. Hum. Genet. 47:776-783, 1990; Fukushi et al., Biochem. Biophys. Res. Comm. 246:613-618, 1998). The ability of TNAP to hydrolyze PPi to Pi (Whyte et al., “Hypophosphatasia,” In: The Metabolic and Molecular Bases of Inherited Disease, ed. Scriver et al., McGraw-Hill Inc., New York, pp. 4095-4112, 1995) has been hypothesized to be central to the ability of TNAP to promote osteoblastic mineralization (Anderson et al., Am. J. Pathol. 151:1555-1561, 1997; Whyte, Endocrine Rev. 15:439-461, 1994, Pizauro et al., Biochim. Biophys. Acta 1368:108-114, 1998). A major action of PPi is to suppress both the deposition and propagation of hydroxyapatite crystals in vitro (Johnson et al., J. Bone Miner. Res. 14:883-892, 1999; Johnson et al., Arthritis Rheum. 42:1986-1997, 1999; Meyer, Arch. Biochem. Biophys. 231:1-8, 1984). Thus, critically timed removal or exclusion of PPi at sites of mineralization appears to be necessary for active crystal deposition to proceed (Johnson et al., J. Bone Miner. Res. 14:883-892, 1999; Johnson et al., Arthritis Rheum. 42:1986-1997, 1999; Meyer, Arch. Biochem. Biophys. 231:1-8, 1984). Since TNAP functions as a PPi-ase in vitro (Moss et al., Biochem. J. 102:53-57, 1967; Rezende et al., Biochem. J. 301:517-522, 1994), the finding that NTPPPH activity is normal in fibroblasts from hypophosphatasia patients further supported the hypothesis that accumulation of PPi in this disease is the result of defective degradation (Caswell et al., J. Clin. Endocrinol. Metab. 63:1237-1241, 1986). In vitro studies have shown that PPi promotes formation of amorphous calcium phosphate, while the subsequent transformation into hydroxyapatite and growth of hydroxyapatite crystals are inhibited (Caswell et al., Crit. Rev. Clin. Lab. Sci. 28:175-232, 1991). Plasma Cell Membrane Glycoprotein-1 (PC-1) is a nucleotide triphosphate pyrophosphate hydrolase (NTPPPH) isozyme expressed by cultured osteoblastic cells (Goding et al., Immunol. Reviews 161:11-26, 1998; Johnson et al., J. Bone Miner. Res. 14:883-892, 1999; Johnson et al., Arthritis Rheum. 42:1986-1997, 1999; Solan et al., J. Bone Miner. Res. 11:183-192, 1996). NTPPPH activity is a property of several members of a phosphodiesterase nucleotide pyrophosphatase (PDNP) family of ecto-enzymes that also includes B10 and autotaxin (Johnson et al., J. Bone Miner. Res. 14:883-892, 1999; Johnson et al., Arthritis Rheum. 42:1986-1997, 1999). PC-1 expression, and the extent of PC-1 distribution to MVs, are regulated by certain growth factors and calciotropic hormones, including TGFα, bFGF, and 1,25 dihydroxyvitamin D3 (Bonewald et al., Bone and Mineral 17:139-144, 1992; Johnson et al., J. Bone Miner. Res. 14:883-892, 1999; Johnson et al., Arthritis Rheum. 42:1986-1997, 1999; Oyajobi et al., J. Bone Miner. Res. 9:99-109, 1994; Oyajobi et al., J. Bone Miner. Res. 9:1259-1269, 1994; Solan et al., J. Bone Miner. Res. 11:183-192, 1996). Osteoblast-derived MV PC-1 appears to function directly to increase MV fraction PPi content and to restrain mineralization by isolated MVs in vitro (Johnson et al., J. Bone Miner. Res. 14:883-892, 1999; Johnson et al., Arthritis Rheum. 42:1986-1997, 1999). In this regard, a two- to four-fold increase in osteoblast PC-1 expression decreases, by greater than 80%, the amount of hydroxyapatite deposited in the pericellular matrix of osteoblasts in vitro (Johnson et al., J. Bone Miner. Res. 14:883-892, 1999; Johnson et al., Arthritis Rheum. 42:1986-1997, 1999). On the other hand a dysregulated increase in chondrocyte PPi production is a central feature of idiopathic chondrocalcinosis (or primary calcium pyrophosphate dihydrate, CPPD, crystal deposition disease) whose prevalence appears to be greater than 15% at age 65 and rises progressively with age. Mean cartilage PPi-generating NTPPPH activity doubles, promoting PPi supersaturation that stimulates CPPD crystal deposition in the pericellular matrix of chondrocytes in articular cartilage and fibrocartilaginous menisci. Interestingly, it appears that both up-regulation as well as inactivation of PC-1 leads to osteoarthitic disease albeit by different molecular mechanisms. The role of PC-1 on mineralization have been confirmed to be physiologically significant in ttw/ttw (formerly known as “tiptoe walking Yoshimura”) mice (Okawa et al., Nature Genet. 19:271(1998), which are homozygous for a naturally occurring PC-1 truncation mutation. In early life, ttw/ttw mice develop not only progressive ossification of spinal and peripheral joint ligaments but also articular and meniscal cartilage calcification.
The ankylosis protein (ANK) has a role in suppressing mineralization by contributing to the extracellular supply of PPi. However, unlike PC-1, ANK appears to function as a transmembrane PPi-channeling protein, allowing PPi molecules to passage through the plasma membrane from the cytoplasm to the outside of the cell (Ho et al., Science 289: 265-269, 2000; Nümberg et al., Nat. Genet. 28: 37-41, 2001). ANK protein is detectable in many tissues, yet its expression is particularly strong in the cartilage of joints (Ho et al., Science 289: 265-269, 2000). Cell surfaces of osteoblasts and chondrocytes appear to be abundant in ANK protein, but in contrast to PC-1 and TNAP, it is not present in the membranes of MVs. The discovery of the ANK protein was recently accomplished by identifying the gene in a naturally occurring mutant mouse strain that had characteristics of progressive ankylosis, thereupon the designation ank mice (Ho et al., Science 289: 265-269, 2000). The ANK gene in this mutant mouse line has a nonsense mutation in the open reading frame that translates into a premature stop codon and a truncated non-functional protein. ank/ank mice develop hydroxyapatite crystals in articular surfaces and synovial fluids. ank/ank mice display pathological abnormalities that mimic several arthritic diseases, including ectopic calcification, cartilage erosion and osteophyte formation seen in osteoarthritis, and vertebral fusion observed in ankylosis spondylitis patients (Sweet and Green, J. Hered. 72: 87-93, 1981; Hakim et al., Arthr. Rheum. 27: 1411-1420, 1984; Sampson and Davis, Spine 13: 650-654, 1988; Mahowald et al., J. Rheumatol. 16: 60-66, 1989).
The clinical linkages of increased PPi production and NTPPPH activity to chondrocalcinosis are well-recognized. Moreover, the association of both PC-1 deficiency and defective ANK function with articular cartilage degeneration and apatite deposition in mice further links basic studies of PC-1 and ANK function and PPi metabolism to clinical arthritic disease. The perispinal ligamentous ossification of both PC-1 deficient and ank/ank mice is noteworthy, because it is modulated by unrestrained growth, and by chondrocytic and osteoblastic metaplasia of perispinal ligament fibroblasts and of periosteum, respectively (Sali et al., “Germline deletion of the nucleoside triphosphosphate (NTPPPH) plasma cell membrane glycoprotein (PC-1) produces abnormal calcification of periarticular tissues,” In: Conference Proceedings: Second International Symposium on Ecto-ATPases and Related Ectonucleotidases; ed. Vanduffel and Lemmens, Shaker Publishing BV, Maastricht, Netherlands, pp. 267-282, 1999; Okawa et al., Nature Gen. 19:271-273, 1998; Ho et al., Science 289:265-270, 2000; Krug et al., J. Rheumatol. 27:1257-1259, 2000).
Osteopontin (OPN), first identified in bone matrix and termed bone sialoprotein 1, is secreted by a wide variety of cell types including osteoblasts and osteoclasts and has the potential to serve as a bridge between cells and hydroxyapatite crystals through RGD and polyaspartic acid motifs (Oldberg et al., Proc. Natl. Acad. Sci. USA 83: 8819-8823, 1986). Despite the broad range of potential functions for OPN, ablation of the OPN gene in mice has not revealed an obvious phenotype that can be clearly connected to a specific function (Liaw et al., J. Clin. Invest. 101: 1468-1478, 1998; Rittling et al., J. Bone Min. Res. 13: 1101-1111, 1998). Opn KO mice demonstrate subtle changes in bone mineralization, such as greater hydroxyapatite crystal size and crystallinity (Boskey et al., Calcif. Tissue Int. 71: 145-154, 2002).